Mutant recombinant hemoglobins containing heme pocket mutations

ABSTRACT

This invention relates to recombinant hemoglobins containing mutations in or near the heme pocket of the hemoglobin molecule. This invention particularly relates to recombinant hemoglobins that have altered geometry or polarity around the distal portion of the heme pocket.

This application is a continuation-in-part of Hoffman and Nagai, Ser. No. 08/158,483, filed Nov. 29, 1993, now U.S. Pat. No. 5,449,759 which is a continuation of Hoffman and Nagai, Ser. No. 07/443,950, filed Dec. 1, 1989, now abandoned, which is a continuation in part of Hoffman and Nagai, Ser. No. 07/194,338, filed May 16, 1988, now U.S. Pat. No. 5,028,588, all owned by Somatogen, Inc.

FIELD OF THE INVENTION

This invention relates to mutant recombinant hemoglobins containing mutations around one or more of the heme pockets of the hemoglobin molecule. The invention relates particularly to mutant recombinant hemoglobins that have altered geometry or polarity around the distal portion of the heme pocket which results in mutant recombinant hemoglobins with reduced autooxidation rates, reduced or increased affinities for ligands or both reduced autooxidation and changed ligand affinity.

BACKGROUND OF THE INVENTION

Loss of blood often requires replacement of both the volume of fluid that is lost and the oxygen carrying capacity of that fluid. This is typically accomplished by transfusing red blood cells, either as packed RBC's or as units of whole blood. However, it is not always possible, practical or desirable to transfuse a patient with donated blood. Human blood transfusions are associated with many risks and limitations, such as:

1) Infectious disease transmission (i.e., human immunodeficiency virus (HIV), non-A and non-B hepatitis, hepatitis B, Yersinia enterocolitica, cytomegalovirus, human T-cell leukemia virus 1)

2) Immunologic reaction (i.e., hemolytic transfusion reaction, immunosuppresion, graft versus host reaction)

3) Typing and cross-matching required prior to administration

4) Limited availability

5) Limited stability (shelf life of 42 days or less; cannot be frozen)

When human blood is not available or the risk of transfusion is too great, volume can be replaced utilizing plasma expanders such as colloid and crystalloid solutions but to date, none of the volume replacement therapies currently approved for human use can transport oxygen. In situations where replacement of lost blood is necessary and blood is not available for transfusion, a red blood cell substitute that can transport oxygen, such as a hemoglobin solution, is desirable. Administration of a hemoglobin solution can increase and/or maintain plasma volume and decrease blood viscosity in the same manner as conventional plasma expanders, but, in addition, a hemoglobin-based red blood cell substitute should be able to support adequate transport of oxygen from the lungs to peripheral tissues. Moreover, an oxygen-transporting hemoglobin-based solution may be used in most situations where red blood cells are currently utilized. For example, oxygen-transporting hemoglobin-based solution may be used to temporarily augment oxygen delivery during or after pre-donation of autologous blood prior to the return of the autologous blood to the patient.

To address this need, a number of red cell substitutes have been developed (Winslow, R. M.(1992) Hemoglobin-based Red Cell Substitutes, The Johns Hopkins University Press, Baltimore 242 pp). These substitutes include synthetic perfluorocarbon solutions, (Long, D. M. European Patent 0307087), stroma-free hemoglobin solutions, both chemically crosslinked and uncrosslinked, derived from a variety of mammalian red blood cells (Rausch, C. and Feola, M., U.S. Pat. Nos. 5,084,558 and 5,296,465; Sehgal, L. R., U.S. Pat. Nos. 4,826,811 and 5,194,590; Vlahakes, G. J. et al., (1990) J. Thorac. Cardiovas. Surg. 100: 379-388) and hemoglobins expressed in and purified from genetically engineered organisms (for example, non-erythrocyte cells such as bacteria and yeast, Hoffman et al., WO 90/13645; bacteria, Fronticelli, C. et al., U.S. Pat. No. 5,239,061; yeast, De Angelo et al., WO 93/08831 and WO 91/16349; and transgenic mammals, Logan et al., WO 92/22646; Townes, T. M and McCune, S. L., WO 92/11283). These red blood cell substitutes have been designed to replace or augment the volume and the oxygen carrying capability of red blood cells.

The oxygen carrying portion of the red blood cell is the protein hemoglobin. Hemoglobin is a tetrameric protein molecule composed of two identical alpha globin subunits (α₁, α₂), two identical beta globin subunits (β₁, β₂) and four heme molecules. A heme molecule is incorporated into each of the alpha and beta globins to give alpha and beta subunits. Heme is a large macrocyclic organic molecule containing an iron atom; each heme can combine reversibly with one ligand molecule such as oxygen. In a hemoglobin tetramer, each alpha subunit is associated with a beta subunit to form two stable alpha/beta dimers, which in turn associate to form the tetramer (a homodimer). The subunits are noncovalently associated through Van der Waals forces, hydrogen bonds and salt bridges.

In the unliganded state (deoxygenated or "deoxy") state, the four subunits form a quaternary structure known as "T" (for "tense") state. During ligand binding, the α₁ β₁ and α₂ β₂ interfaces remain relatively fixed while the α₁ β₂ and α₂ β₁ interfaces exhibit considerable movement When a ligand is bound to the hemoglobin molecule, the intersubunit distances are increased relative to the deoxygenated distances, and the molecule assumes the "relaxed" or "R" quaternary structure which is the thermodynamically stable form of the molecule when three or more ligands are bound to the heme.

Red blood cell replacement solutions have been administered to animals and humans and have exhibited certain adverse events upon administration. These adverse reactions may include hypertension due to vasoconstriction, renal failure, neurotoxicity, and liver toxicity (Winslow, R. M., ibid., Biro, G. P. et al., (1992) Biomat., Art. Cells & Immob. Biotech. 20: 1013-1020) and in the case of perfluorocarbons, hypertension, activation of the reticulo-endothelial system, and complement activation (Reichelt, H. et al., (1992) in Blood Substitutes and Oxygen Carriers, T. M. Chang (ed.), pg. 769-772; Bentley, P. K. ibid, pp. 778-781). For hemoglobin based oxygen carriers, renal failure and renal toxicity is the result of the formation of hemoglobin α/β dimers. The formation of dimers can be prevented by chemically crosslinking (Sehgal, et al., U.S. Pat. Nos. 4,826,811 and 5,194,590; Walder, J. A. US Reissue Patent RE34271) or genetically linking (Hoffman, et al., WO 90/13645) the hemoglobin dimers so that the tetramer is prevented from dissociating.

However, prevention of dimer formation has not alleviated all of the adverse events associated with hemoglobin administration. Blood pressure changes upon administration of hemoglobin solutions have been attributed to vasoconstriction resulting from the binding of endothelium derived relaxing factor (EDRF) by hemoglobin (Spahn, D. R. et al., (1994) Anesth. Analg. 78: 1000-1021; Biro, G. P., (1992) Biomat., Art. Cells & Immob. Biotech., 20: 1013-1020; Vandegriff, K. D. (1992) Biotechnology and Genetic Engineering Reviews, Volume 10: 404-453 M. P. Tombs, Editor, Intercept Ltd., Andover, England). Endothelium derived relaxing factor has been identified as nitric oxide (NO) (Moncada, S. et al., (1991) Pharmacol. Rev. 43: 109-142 for review); both inducible and constitutive NO are primarily produced in the endothelium of the vasculature and act as local modulators of vascular tone. CO has also been implicated in blood pressure regulation since it can also activate guanylate cyclase (Snyder, S. H. and Bredt, D. S. (1992) Sci. American May, 68-77). Hemoglobin can bind both nitric oxide and carbon monoxide, thus preventing vascular relaxation and potentially leading to the hypertension sometimes observed upon administration of extracellular hemoglobin solutions. In addition to direct binding to deoxyhemoglobin, NO can also oxidize oxyhemoglobin producing peroxynitrite and methemoglobin. This reaction could also lower free concentrations of NO and lead to hypertension.

Some inflammatory responses are also mediated by nitric oxide (Vandegriff, ibid., Moncada, S., et al., ibid.). For example, nitric oxide produced by the endothelium inhibits platelet aggregation and as nitric oxide is bound by cell-free hemoglobin solutions, platelet aggregation may be increased. As platelets aggregate, they release potent vasoconstrictor compounds such as thromboxane A₂ and serotonin (Shuman, M. (1992) in Cecil Textbook of Medicine, J. B. Wyngaarden, L. H. Smith and J. C. Bennett, ed., W. B. Saunders Co, Philadelphia, pages 987-992). These may act synergistically with the reduced nitric oxide levels due to binding by hemoglobin to result in an exaggerated vasoconstriction.

In addition to modulating platelet aggregation, nitric oxide inhibits neutrophil attachment to cell walls. Increased adhesion of neutrophils to cell walls may lead to cell wall damage. Endothelial cell wall damage has been observed upon infusion of some hemoglobin solutions; this kind of damage is consistent with uptake of endogenous nitric oxide by hemoglobin (White, et al., (1986) J. Lab. Clin. Med. 108: 121-131; Vandegriff (1992) ibid). In all these cases, a hemoglobin molecule with reduced reactivity for either binding or oxidizing nitric oxide and with a physiologically acceptable oxygen affinity might ameliorate some of these observed adverse events while still functioning as an effective oxygen carrier.

Conversely, while a moderate production of nitric oxide is required to maintain appropriate vascular tone, excess production of nitric oxide by the endothelium and other nitric oxide producing cells, such as macrophages, may result in pathological states. These disease states include septic shock and nitric oxide mediated hypotension. In these cases, binding of excess nitric oxide or its oxidation by a hemoglobin with a particularly high reactivity for nitric oxide may be useful (Kilbourn, PCT Application Number WO 93/16721).

Another disadvantage of hemoglobin-based blood substitutes has been the tendency of the iron in the heme to undergo oxidation. The oxidation of the iron in the heme from the ferrous (Fe⁺²) to the ferric form (Fe⁺³) results in the formation of methemoglobin. Methemoglobin is a non-functional form of hemoglobin that cannot bind oxygen, thus its formation in hemoglobin solutions results in reduced capacity to bind gases and may thus require administration of larger amounts of solution to accomplish the same desired benefit or result. In addition, methemoglobin molecules are vulnerable to accelerated degradation due to hemichrome formation, heme loss, precipitation, and reaction with hydrogen peroxide to form toxic radicals and the like (Bunn, H. F. and Forget, B. G. Hemoglobin: Molecular, Genetic and Clinical Aspects, W. B. Saunders Company, Philadelphia, 690 pp, hereby incorporated by reference; Rachmilewitz, E. A. (1974) Sem. Hematol. 11: 441-462).

Myoglobin is a monomeric heme protein found in muscle which binds oxygen reversibly which has often been used as a simple model for the complex chemistry of tetrameric hemoglobin. Myoglobin appears to be an adequate model for ligand binding to the alpha subunits of hemoglobin. In contrast, it is a less satisfactory model for the ligand binding and oxidative behavior of the beta subunit. Moreover, because myoglobin is a non-cooperative molecule, changes in overall ligand affinity and oxidation are not predictable when a mutation in myoglobin is transferred to the equivalent location in the hemoglobin molecule.

Nevertheless, the autooxidation rate and the discrimination between O₂, CO, and NO in both myoglobin and hemoglobin appear to be the result of polar interactions between bound ligands and the histidine found at the E7 position in both proteins. In unliganded myoglobin and the subunits of hemoglobin, a H₂ O molecule is hydrogen bonded to the histidine and must be displaced before a ligand can bind to the iron atom of the heme. The binding of all three ligands requires displacement of this water molecule. In the case of O₂ binding, the inhibition caused by the displacement of the water molecule is overcome by much more favorable hydrogen bonding interactions between His-E7 and the polar Fe.sup.δ (+)--O--O.sup.δ (-) complex. No favorable interactions occur between bound CO and the histidine at the E7 position. Thus the affinity of hemoglobin for CO is primarily a function of the intrinsic strength of the covalent bond between CO and the heme iron atom, and as a result, the requirement for H₂ 0 displacement results in a net inhibition of CO binding. Without this inhibition, CO would bind approximately 10 fold more tightly to alpha subunits of hemoglobin, and the discrimination against it and for oxygen would be compromised. NO binding shows an intermediate situation; the inhibition due to water displacement is exactly balanced by a weak hydrogen bond with His-E7. As a result, the NO affinity of hemoglobin is similar to that of model heme compounds. Note also that the formation of the iron-oxygen/His E7 complex results in both a net enhancement of oxygen affinity relative to carbon monoxide affinity and a decrease in spontaneous iron oxidation. Thus oxygen affinity and autooxidation are often tightly coupled. In addition, oxidation can occur by an NO mediated mechanism (FIG. 1). NO is a free radical and can react rapidly with reduced iron-oxygen complexes to form peroxynitrite (--OONO) and ferric iron. In hemoglobins and myoglobins, the NO must first diffuse into the heme pocket and take a position in cavity circumscribed by Val(E11), Leu(B10), Phe(CD1), and Leu or Ile(G8) after which reaction with the bound oxygen occurs. Placement of large aromatic residues at these positions should inhibit this oxidative process and prevent NO consumption by oxyhemoglobin.

An effective extracellular hemoglobin blood substitute must bind oxygen cooperatively with moderately low affinity (P₅₀ ≧20-30 mm Hg or 30 to 50 μM free O₂ measured at 37° C.): where the P₅₀ defined as the oxygen partial pressure at half saturation of hemoglobin) and must have large association and dissociation rate constants for oxygen binding to allow efficient uptake in the lungs and delivery in muscle capillaries. The rate and extent of oxygen delivery is proportional to the P₅₀ of the extracellular hemoglobin or red cells present in muscle capillaries (Vandegriff, K. D., and Olson, J. S. (1984) J. Biol. Chem. 259: 12619-12627.; Lemon, D. et al., (1987) J. Appl. Physiol. 62: 798-806; Nair, P. K. et al., (1989) Microvascular Research 38: 269-285). Lowering oxygen affinity results in more efficient O₂ transport and is limited only by the need to achieve 80-90% saturation in the alveolar capillaries. Unfortunately, almost all attempts to raise P₅₀ by chemical modification, mutation, and allosteric effectors cause higher rates of autooxidation and subsequent hemin loss and denaturation. Brantley et al. (Brantley, R. E. Jr., et al., (1993) J. Biol. Chem. 268: 6995-7010) have shown that there is strong inverse correlation between oxygen affinity and the rate of autooxidation, k_(ox), for 27 different recombinant myoglobins. Similar correlations have been noted both anecdotally and experimentally for tetrameric hemoglobins (Vandegriff, K. D. (1992) Biotechnology and Genetic Engineering Reviews 10: 403-453). This is a major stumbling block for engineering more efficient blood substitutes since stability is compromised by elevated P₅₀ values.

The dominant mechanism for autooxidation in myoglobin, and quite likely hemoglobin, involves protonation of the Fe(II)--O₂ complex and dismutation into Fe(III) and the neutral perhydroxyl radical HO₂. The bimolecular reaction of molecular O₂ with deoxymyoglobin containing weakly coordinated water only contributes to the observed rate when the unimolecular Fe(II)--O--O--H.sup.(+) dissociation process is slow, and then only when the O₂ concentration approaches the P₅₀. In native oxymyoglobin, the neutral side chain of His(E7) forms a hydrogen bond with the bound ligand. This interaction increases O₂ affinity and, at the same time, decreases the rate of autooxidation by preventing net protonation of the Fe--O₂ complex since the pK_(a) for forming the imidazolate anion is ≧12.

Based on the physical-chemical mechanisms underlying spontaneous and NO induced autooxidation of the heme in the distal pocket and the binding of ligands at the heme group, the inventors have redesigned the active site in the region directly adjacent to the heme iron atom to moderate both the oxidation rate and ligand affinity. The present inventors have been able to develop strategies for selectively altering the specificity of recombinant heme proteins for the three physiologically important gases, O₂, CO, and NO, and for decoupling the rate of oxidation of the iron atom from affinity for gaseous ligands. The present inventors have discovered that if the distal residue histidine at position E7 of either the alpha or the beta globin is replaced with certain other amino acids, the affinity of the resultant hemoglobin mutant for both NO and CO can be increased. Likewise, replacement of leucine at the B10 helical position of the alpha or beta globin can result in a hemoglobin that shows high affinity for NO without a concomittant increase in affinity for CO. In addition, the inventors have also discovered that there are mutations of the distal heme pocket that inhibit dramatically the reactions of NO and CO with hemoglobin. These mutations include replacements of Leu-B10, Val-E11, and His-E7, and various multiple combinations of these mutations, particularly double and triple combinations. Lastly, the present inventors have discovered that to protect against protonation by solvent water and subsequent oxidation of the iron, the spaces adjacent to the bound oxygen can be filled with large aromatic residues. These aromatic groups exclude water by their steric effects and partially stabilize the polar iron-oxygen complex by interactions between the phenyl ring electronic multipole and bound oxygen. The size (the free volume) of the distal pocket can be decreased by introducing these and other mutations, and this decrease in size of the distal pocket also excludes other exogenous oxidizing agents such as NO and H₂ O₂ from direct reaction with bound O₂ (FIG. 1).

Genetic engineering techniques have allowed the expression of heterologous proteins in a number of biological expression systems, such as insect cells, plant cells, transgenic cells, yeast systems and bacterial systems. Because the sequences of alpha and beta globin of hemoglobin are known, and efficient expression criteria have been determined, it is possible that any suitable biological protein expression system can be utilized to produce large quantities of mutant recombinant hemoglobin. Indeed, hemoglobin has been expressed in a number of biological systems, including bacteria (Hoffman et al., WO 90/13645), yeast (De Angelo et al., WO 93/08831 and WO 91/16349; Hoffman et al., WO 90/13645) and transgenic mammals (Logan et al., WO 92/22646; Townes, T. M and McCune, S. L., WO 92/11283).

Mutants of hemoglobin are known and disclosed in PCT publication number WO88/09179, hereby incorporated by reference. Brief reviews of the effects of some distal pocket mutations on ligand binding to myoglobin and hemoglobin have been presented by Perutz (Perutz, M. F. (1989) Trends Biochem. Sci. 14: 42-44); Springer et al. (Springer, B. A., et al. (1994) Chem. Rev. 94: 699-714) and Mathews et al. (Mathews, A. J. et al., (1989) J. Biol. Chem. 264: 16573-16583) and the differences between myoglobin and hemoglobin have been noted in these and other publications. Both the alpha and beta globin subunits have been sequenced (Hoffman and Nagai, U.S. Pat. No. 5,028,588, hereby incorporated by reference) and techniques for the mutation, expression and purification of the mutant recombinant hemoglobins have been described (Looker, D. et al. (1994) Expression of Recombinant Hemoglobin in Escherichia coli In: Methods in Enzymology 231: 364-374, S. O. Colowick, ed.; Academic Press, Inc.; Hoffman et al., WO 93/13645; Milne et al., co-pending U.S. application, Ser. No. 08/339,304, filed Nov. 14, 1994).

SUMMARY OF THE INVENTION

This invention relates to mutant recombinant hemoglobins containing mutations in or near the heme pocket of the hemoglobin molecule. More particularly, this invention relates to mutant recombinant hemoglobins that have altered geometry or polarity around the distal portion of the heme pocket.

Further, the present invention relates to mutant recombinant hemoglobin molecules containing heme pocket mutations wherein the mutations result in a mutant recombinant hemoglobin having autooxidation rates lower than autooxidation rates measured for naturally occurring purified human hemoglobin under comparable conditions. Representative examples of mutations that result in such reduced autooxidation rate hemoglobin mutants include:

Leu-B10→Phe

Val-E11→Phe

both His-E7→Gln and Val-E11→Phe

both His-E7→Gln and Leu-B10→Phe

His-E7→Gln, Leu-B10→Phe and Val-E11→Phe

and

His-E7→Phe, Leu-B10→Phe and Val-E11→Phe.

The present invention also relates to mutant recombinant hemoglobin molecules containing heme pocket mutations wherein the mutations result in a mutant recombinant hemoglobin having an affinity for gaseous ligands greater than or less than purified natural human hemoglobin measured under the same conditions. Preferably, the gaseous ligands bind to the hemoglobin at the heme pocket of the hemoglobin molecule and more preferably, the gaseous ligands are selected from the group consisting of carbon monoxide and nitric oxide, most preferably the gaseous ligand is nitric oxide. The mutant recombinant hemoglobin molecules of the present invention preferably contain mutations that result in alteration of the amino acid composition of distal pocket so that the distal pocket amino acid composition no longer matches the amino acid composition of human hemoglobin. Representative examples of mutations that result in mutant recombinant hemoglobins with altered affinity for gaseous ligands include the following site specific mutations:

Leu-B10→Trp

Leu-B10→Phe

Leu-B10→Val

Leu-B10→Ile

Leu-B10→Ala

His-E7→Leu

His-E7→Trp

and

Val-E11→Phe

Val-E11→Trp

It is also within the scope of this invention to provide methods for the preparation of mutant recombinant hemoglobins with altered affinities and/or oxidation properties by appropriate mutation of the alpha and/or beta globin gene, expression and purification of the mutant recombinant hemoglobin. Methods for selecting candidate mutations and evaluating their ligand affinities are described herein. The invention is further directed to mutant recombinant hemoglobins containing the above described mutations in the alpha subunit, the beta subunit, or both the alpha and beta subunits and having altered autooxidation or affinity for gaseous ligands, preferably having altered affinity for nitric oxide or carbon monoxide. The invention further relates to DNA's or vectors containing DNA sequences coding for the mutant recombinant hemoglobins of the invention.

The invention further relates to pharmaceutical compositions containing the mutant recombinant hemoglobins of the invention.

Specific embodiments of the present invention include hemoglobins with both unusually high and low affinities for carbon monoxide or nitric oxide. In another embodiment of the invention, hemoglobins are provided which are recombinant oxyhemoglobins with reduced affinity for NO and decreased NO and H₂ O₂ mediated oxidation relative to naturally occurring human hemoglobins.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof and from the claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. Unless mentioned otherwise, the techniques employed or contemplated herein are standard methodologies well known to one of ordinary skill in the art.

For the purposes of this invention, "naturally occurring human hemoglobin", "native hemoglobin" or "conventional hemoglobin" refer to the species of hemoglobin A whose alpha and beta chains amino acid sequences are the same as the amino acid sequences given in SEQ ID.:1: and SEQ ID.:2: respectively. Note that it is conventional to identify the helical segments of the globin subunits by letters, for example, the proximal histidine of the alpha chain or the beta chain is termed F8 (residue 8 of helix F). The non-helical segments are identified by letter pairs, indicating which helical segments they connect, for example, non-helical segment BC connects helix B and helix C. The helical notation and corresponding amino acids for alpha and beta globin are shown in Table 1.

"Recombinant hemoglobin" means hemoglobin, whether native or mutant, comprising alpha-like globin proteins and/or beta-like globin proteins, at least one of which is obtained by expression of a globin gene carried by a recombinant DNA molecule in a cell other than the cell in which that hemoglobin gene and/or hemoglobin protein is naturally found, i.e., the hemoglobin gene is heterologous to the host in which it is expressed. Therefore, the expression of any human hemoglobin gene in any cell other than a human red blood cell would be considered to be a recombinant hemoglobin. Moreover, the expression of a vertebrate hemoglobin in any species of invertebrate, or any vertebrate other than the vertebrate where the hemoglobin to be expressed is naturally occurring, would be considered a recombinant hemoglobin. The expression of any naturally occurring hemoglobin mutant in any species other than the species in which it is naturally occurring, would be considered a recombinant hemoglobin. The expression of any non-naturally occurring mutant hemoglobin in any species would be considered a recombinant hemoglobin. The expression of a naturally occurring mutant hemoglobin in any individual organism, regardless of species, other than the individual organism in which said mutant is naturally expressed would be considered a recombinant hemoglobin.

An "alpha-like globin" (or domains thereof) has at least about 75% sequence identity with native human alpha globin. However, a polypeptide of lesser sequence identity may still be considered substantially homologous with alpha globin, and thus may be an alpha-like globin, if it has a greater sequence identity than would be expected from chance and also has the characteristic higher structure of alpha globin and similar biological activity. Likewise, a "beta-like globin" (or domains thereof) has at least about 75% sequence identity with native human beta globin. However, a polypeptide of lesser sequence identity may still be considered substantially homologous with beta globin, and thus may be a beta-like globin, if it has a greater sequence identity than would be expected from chance and also has the characteristic higher structure of beta globin and similar biological activity.

"Liganded hemoglobin" means hemoglobin to which a ligand is bound at the heme groups. Common preferred ligands include, but are not limited to O₂, CO, NO and the like.

"Oxyhemoglobin" means hemoglobin in which each of the functional oxygen binding sites has bound to it an oxygen molecule.

"Deoxyhemoglobin" or "unliganded hemoglobin" means any hemoglobin to which no ligand is bound to the alpha globin, the beta globin, and/or any functional heme prosthetic group.

"Methemoglobin" or "oxidized hemoglobin" means any hemoglobin in which the iron has been oxidized to the ferric state.

"R-state hemoglobin" is the high affinity state of hemoglobin and is the dominant form of hemoglobin when a ligand is bound at the heme pockets. The ligand is typically oxygen, thus this state is known as the "oxy" or "R" (for relaxed) state. In the R state, intersubunit distances are increased relative to the distances in T-state hemoglobin.

"T-state hemoglobin" is the low affinity state of hemoglobin and is the dominant form of hemoglobin when it is deoxygenated ("deoxy", or "T" for "tense").

"Heme pocket" means that pocket formed around the heme of each globin subunit described by the residues and is meant to include residues within about 6 Å of the heme pocket.

"Distal heme pocket" means that portion of the heme pocket above the plane of the heme that contains the free coordination site of the iron where ligand molecules can combine reversibly with the iron atom and which contains such residues as histidine E7 and valine E11. Likewise, the proximal side of the heme pocket is described by those residues below the plane of the heme and contains such residues as the proximal histidine at position F8.

"Oxidation" means the oxidation of the iron in the heme of any or all of the subunits making up the hemoglobin tetramer from the ferrous (Fe⁺²) to the ferric form (Fe⁺³). Autooxidation occurs spontaneously without the addition of exogenous oxidants, however oxidation can be induced by the presence of exogenous oxidizing agents, most notably NO and hydrogen peroxide.

"Mutations" are substitutions, deletions or additions of one or more amino acids to the amino acid sequence that constitutes naturally occurring human hemoglobin.

"Affinity" is the overall binding of a ligand to hemoglobin, and is described by the thermodynamic equilibrium constant, Keq. Affinity is the ratio of the ligand association rate and the hemoglobin--ligand dissociation rate constants, and thus changes in either rate constants can lead to changes in ligand affinity.

"Altered affinity" means the affinity of a recombinant hemoglobin for a gaseous ligand that is at least 10% different from the affinity of naturally occurring human hemoglobin for that same gaseous ligand under the same measurement conditions.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the proposed mechanism for the reaction of NO with oxyhemoglobin.

FIG. 2 shows schematic diagrams of several key distal pocket mutations which are part of the invention described in this application.

FIG. 2A shows the Leu(B10) to Phe substitution.

FIG. 2B shows the His(E7) to Gln mutation

FIG. 2C shows the double mutant containing both the Leu(B10) to Phe substitution and the His(E7) to Gln mutation, which provides optimum oxygen binding and low autooxidation.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to mutant recombinant hemoglobins containing mutations around the heme pocket of one or more of the globin subunits. Heme is the prosthetic group of hemoglobin, myoglobin, catalase, peroxidase, and cytochrome b. The heme is inserted in a cleft between the E and F helices. The heme iron is linked covalently to the imidazole nitrogen of the "proximal" F8 histidine. The distal E7 histidine and E11 valine appear to guard the access of oxygen to the heme pocket. The residues of the heme pocket include those residues that are on a nearest atom-to-nearest atom basis within 6 angstroms of the heme moiety, more preferable within 4 angstroms of the heme moiety (Fermi, et al. (1984) J. Mol. Biol. 175: 159-174). These residues include for alpha globins:

    ______________________________________                                         First shell           Second Shell                                             ______________________________________                                         Distal residues:                                                               B10 Leu               B13 Met                                                  CE1 Phe               CE3 His                                                  E7 His                CE4 Phe                                                  E11 Val               E10 Lys                                                  G8 Leu                E14 Ala                                                                        G12 Leu                                                  Proximal residues:                                                             F8 His                C7 Tyr                                                                         F4 Leu                                                                         F7 Leu                                                                         FG3 Leu                                                                        FG5 Val                                                                        G4 Asn                                                                         G5 Phe                                                                         H15 Val                                                                        H19 Leu                                                  and for beta globin:                                                           Distal residues:                                                               B10 Leu               B13 Leu                                                  CD1 Phe               CD3 Ser                                                  E7 His                CD4 Phe                                                  E11 Val               E10 Lys                                                  G8 Leu                E14 Ma                                                                         G12 Leu                                                  Proximal residues:                                                             F8 His                C7 Phe                                                                         F4 Leu                                                                         F7 Leu                                                                         FG3 Leu                                                                        FG5 Val                                                                        G4 Asn                                                                         G5 Phe                                                                         G12 Leu                                                                        H15 Val                                                                        H19 Leu                                                  ______________________________________                                    

Note that first shell residues are those residues in close or direct contact with the heme iron atom and/or the bound ligand, while second shell residues are those amino acids which are not in direct contact with the heme or the bound ligand but that interact directly with first shell residues. The invention also relates to mutant hemoglobins containing mutations in any of the residues noted above, as well as double, triple and higher multiple mutations.

As noted above, the protein and DNA sequences of naturally occurring human hemoglobin are known. The present inventors have recognized and identified the mechanism by which not only ligand binding but also susceptibility to oxidation can be modulated by alteration of the residues at the heme pocket. The inventors have particularly identified the regions of the hemoglobin molecule that lead to reduced autooxidation and/or altered ligand binding properties. Moreover, the present inventors have discovered that certain mutations in the region of the heme pocket of hemoglobin result in selective increases or decreases in the affinity of one ligand without a concomittant change in the affinity of other ligands. By the same token, the present inventors have discovered mutations in the same region of the heme that result in hemoglobins that are less susceptible to autooxidation but that nonetheless retain oxygen ligand binding properties that would allow their use as oxygen carriers.

Hemoglobin mutants with high affinities for carbon monoxide and nitric oxide can be used to bind excess nitric oxide or carbon monoxide in vivo for example, in cases of carbon monoxide poisoning or in disease states characterized by excess nitric oxide. In addition, such high affinity hemoglobin mutants can be used to extract nitric oxide or carbon monoxide from cell culture or solution, or can be used to measure the rate of dissociation of these gaseous ligands from other protein molecules. Hemoglobins with low affinities for nitric oxide can be used as blood substitutes with reduced side effects due to reduced binding of nitric oxide. Mutant hemoglobins with low or no affinity for ligands can be used as inert drug delivery vehicles or in non-oxygen delivery applications of hemoglobin, such as therapeutics to enhance hematopoiesis. Hemoglobin mutants that are less susceptible to autooxidation can be used in all applications where hemoglobin can be used, for example as blood substitutes, in hemoaugmentation, and for the treatment of anemias. These more stable hemoglobin mutants can also be used as stable reagents to calibrate hemoglobin measurement instrumentation in clinical applications.

Val-E11 and His-E7 are highly conserved residues which are in Van der Waals contact with the oxygen molecule liganded to the heme iron atoms of hemoglobin; by replacing these residues the intrinsic oxygen affinity of hemoglobin can be altered. Val-E11 has been replaced with Ile, Leu, Ala, Phe and Met The oxygen affinity of the Ala-E11 beta mutant was higher than that of naturally occurring human hemoglobin; that of the Ile-E11 beta mutant was lower. X-ray crystallographic study of the latter mutant showed that the delta-methyl group of the Ile side chain must be pushed to one side if oxygen is to bind to the iron atom. Another alteration that we have made is beta His(E7)→Phe. This mutant has an extraordinarily low oxygen affinity.

In general, mutations around the heme-O₂ binding site that interfere with O₂ binding are also desirable because of their low-affinity O₂ binding nature. Replacing residues that are adjacent to the face of heme that binds O₂ can result in lower affinity. A naturally occurring mutant Hemoglobin Bristol (beta(E11) Val→Asp) has been described with low affinity. Other mutants that are desirable are the beta ile(E11) described herein, beta Asp(E11) and beta Glu(E11). Other residues are also in the vicinity of the O₂ binding site. Histidine E7 (beta His⁶³) can be replaced with Phe, which results in very low O₂ affinity. The other likely residue for mutation is beta Phe(CD1); replacement with Trp is likely to result in low O₂ affinity. The corresponding residues of the alpha chain may be altered instead of, or in addition to, these preferred mutations of the beta chain.

In order to increase CO affinity, His-E7 is replaced with Leu causing an apolar ligand binding site and the loss of internal water in the unliganded form. As a result, CO does not have to displace water and binds extremely tightly with an affinity that can be 30 times greater than that for His-E7 containing protein. This replacement also increases NO affinity for the same reason. However, O₂ affinity is greatly reduced due to the lack of stabilizing hydrogen bonding interactions. Thus, the Leu-E7 mutation can be used to engineer a hemoglobin with an extremely high affinity for CO. The resultant protein can be used to "soak" up CO from the red cells of patients with carbon monoxide poisoning and to measure the amounts of this gas generated by heme oxygenase in cell suspensions or capillaries.

Selective increases in NO and O₂ affinity can be achieved by replacing Leu-B10 with Phe, which displaces distal pocket water due its larger size and at the same time stabilizes both bound NO and O₂ by favorable electrostatic interactions with the positive edge of the phenyl ring (FIG. 5A). The resultant mutant protein shows a 6-fold increase in selectivity of NO over CO and a 10-fold selectivity of O₂ over CO. In addition, the spontaneous and chemically induced oxidations of this mutant are inhibited due to the markedly decreased size of the ligand binding pocket. This mutation in hemoglobin can be used to measure NO levels in plasma and to elevate blood pressure by removing NO generated by NO synthase in endothelial cells.

The Leu-B10 to Trp mutation inhibits markedly the binding of all three ligands. Not only are the affinities decreased 10 to 100-fold but the rates of binding are decreased to an even greater extent. The large indole side chain prevents access to the iron atom. This effect can be made even greater by incorporating large residues at the E11 position. The Trp-B10 single and double mutants interact poorly with all gases and become, in effect, inert hemoglobins and myoglobins. This allows their use as controls for measuring the effects of the proteins themselves and as vehicles for the transport of other drugs in the circulatory system without interfering with the metabolism of either NO or CO.

It has therefore been shown that the affinity of hemoglobin for gaseous ligands, particularly nitric oxide, carbon monoxide and oxygen can be altered at will by replacing residues near the ligand binding site. By adjusting ligand affinity in this way the efficiency of gas transport or gas uptake and release can be optimized to suit a specific application of the mutant recombinant hemoglobin molecule in the absence of allosteric effectors such as 2,3-DPG.

Mutations of beta residues 28 (B10), 42 (CD1), 45 (CD4), 63 (E7), 67 (E11) and 70 (E14) are of particular interest. Other beta residues of interest include 31 (B13), 44 (CD3), 66 (E10), 106 (G8) and 110 (G12). Alpha residues of interest include 29 (B10), 32(B13), 43 (CE1), 45 (CE3), 46 (CE4), 58 (E7), 61 (E10), 62 (E11), 101 (G8) and 105 (G12).

The amino acids in the immediate vicinity of bound O₂ can be mutated to residues which inhibit both spontaneous and chemically induced oxidation of the iron atom and at the same time raise the partial pressure of oxygen required to achieve 50% saturation (the P₅₀ value). In oxyhemoglobin a chemical oxidant (for example, NO) usually first diffuses into the heme pocket and take a position in the cavity circumscribed by Val(E11), Leu(B10), Phe(CD1), and Leu or Ile(G8) after which reaction with the bound oxygen occurs. Placement of large aromatic residues at these positions should inhibit this oxidative process and thus prevents NO consumption by oxyhemoglobin. The mutations can be in either the α and β chains of recombinant hemoglobin or both the α and β chains and include but are not limited to: (1) Leu-B10→Phe or Trp; (2) Val-E11→Phe or Trp; (3) Leu or Met (B13)→Phe or Trp; (4) Leu (G8)→Phe or Trp; (5) His-E7→Gln/Val-E11→Phe; (6) His-E7→Gln/Leu-B10→Phe; (7) His-E7→Gln/Leu-B10→Phe/Val-E 11→Phe; and (8) His-E7→Phe/Leu-B10→Phe/Val-E11→Phe and other multiple mutations at the primary and secondary shells of the distal pocket.

Any of the mutations of the amino acid in the hemoglobin sequence described herein can be accomplished by a number of methods that are known in the art. Mutation can occur at either the amino acid level by chemical modification of an amino acid or at the codon level by alteration of the nucleotide sequence that codes for a given amino acid. Substitution of an amino acid at any given position in a protein can be achieved by altering the codon that codes for that amino acid. This can be accomplished by site directed mutagenesis using: (1) the Amersham technique (Amersham mutagenesis kit, Amersham, Inc., Cleveland, Ohio) based on the methods of Taylor et al., Nucl. Acids Res. (1985) 13: 8749-8764; Taylor et al., (1985) Nucl. Acids Res. 13: 8764-8785; Nakamaye and Eckstein, (1986) Nucl. Acids Res. 14: 9679-9698; and Dente et al., in DNA Cloning, Glover, Ed., IRL Press (1985) pages 791-802, (2) the Promega kit (Promega Inc., Madison, Wis.) or (3) the Biorad kit (Biorad Inc., Richmond, Calif.), based on the methods of Kunkel, (1985) Proc. Natl. Acad. Sci. USA 82: 488; Kunkel et al., (1987) Meth. Enzymol. 154: 367; Kunkel, U.S Pat. No. 4,873,192. It can also be accomplished by other commercially available or non-commercial means which incorporate the technique of site-directed mutagenesis (using mutant oligonucleotides to achieve mutagenesis).

Site directed mutagenesis can also be accomplished using PCR based mutagenesis such as that described in Zhengbin et al., pages 205-207 in PCR Methods and Applications, Cold Spring Harbor Laboratory Press, New York (1992); Jones and Howard, (1990) BioTechniques 8(2): 178; Jones and Howard, (1991) BioTechniques 10: 62-66.

Site directed mutagenesis can also be accomplished using cassette mutagenesis with techniques that are known to those of skill in the art.

Suitable pharmaceutical compositions for the mutant recombinant hemoglobins of the invention are described in application Milne, et al., Ser. No. 08/339,304, filed Nov. 14, 1995, now abandoned, and Caspari et al., Method for Facilitating the Use of Autologous Blood to Replace Lost Blood, filed Jan 13, 1995.

The foregoing description of the specific embodiments reveal the general nature of the invention so that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation.

All references cited herein are hereby incorporated by reference for their relevant teachings.

EXAMPLES

The following examples are provided by way of describing specific embodiments of the present invention without intending to limit the scope of the invention in any way.

Example 1 Production of Mutant Recombinant Hemoglobins

A. Construction of a Bacterial System for the Production of Mutant Recombinant Hemoglobins

Mutant recombinant hemoglobins were produced by fermentation of the E. coli strain 753 (described in co-pending application Ser. No. 08/188,374, filed Jan. 27, 1994) carrying either the plasmid pSGE0.0E4 or pSGE1.1E4. Plasmid pSGE0.0E4 codes for wild type recombinant human hemoglobin while pSGE1.1E4 codes for recombinant hemoglobin genetically linked by a glycine linker between the C terminus of a first alpha chain and the N terminus of a second alpha chain. Construction of the plasmids are described below and in PCT/US90/02654 and application Hoffman et al., U.S. Ser. No. 07/789,179 filed Nov. 8, 1991 now U.S. Pat. No. 5,545,727.

The αL29F (αLeuB10→Phe) mutation was constructed using oligonucleotide site-directed mutagenesis in the M13 vector phagescript according to the procedure described by Zoller and Smith (Zoller, M. J. and Smith, M. (1987) Meth. Enz. 100: 468-500) Mutants were screened by their ability to differentially hybridize to the radiolabelled mutagenic oligonucleotide at high temperature and low salt conditions. Once the mutants were identified, mutants were then sequenced to confirm the presence of the mutation. The mutated alpha gene was then subcloned back into pSGE0.0E4. If the mutant alpha gene was placed into pSGE1.1E4, three subcloning steps were required:

1) Transfer of the mutant alpha gene from phagescript as an Eag1-Pst1 fragment into the Eag 1-Pst1 digested desval alpha pGem vector. This step provided the mutant alpha gene with the correct 5' terminus.

2) A mutant dialpha gene with the mutations in the 3' alpha gene was constructed by inserting the Eag1 DNA fragment from dialpha pGem into the Eag1 site of the relevant mutant desval alpha pGem plasmid.

3)Finally each of the mutant dialpha genes were cloned into the pSGE1.1E4 expression vector as a Sma1-Pst1 fragment.

Transformations into DH5α Hoffman et al., U.S. Pat. No. 5,545,727 issued Aug. 13, 1996), now U.S. Pat. No. 5,545,721 at each step in the subcloning procedure were carried out according to standard protocols (Maniatis et al. (1982) Molecular Cloning Cold Spring Harbor, N.Y.). The presence of the relevant mutation in the correct a gene was confirmed by sequencing at each stage in the subcloning procedure.

The βL28F (βLeu B10→Phe) mutation was made by cassette mutagenesis of the wild type β gene. In this particular case, complementary oligonucleotides which spanned the region between the Sac II and Bgl II sites of the β gene were synthesized. These oligonucleotides incorporated the βL28F mutation. The mutagenesis was carried out in the pGEM1/wild type β plasmid and the mutant β gene was then subcloned back into the pSGE0.0E4 vector as a Pst I-Bgl II fragment.

Mutant hemoglobins containing the Leu B10→Phe mutation in both the alpha and beta chains were constructed by replacing the wild type beta with the mutant beta gene into the subclone that already contained the alpha mutation.

B. Fermentations

Fermentor Inoculum (500 mL Broth in 2 L Shake Flasks)

To prepare the fermentor inoculum, seed stock was thawed. Seed stock (100 ml) was grown up in 500 ml of DM1 in an Erlenmeyer flask at 37° C. in s 1 inch rotary shaker (275 to 300 rpm) for 8 to 10 hours. DM1 media is:

4.1 g/L KH₂ PO₄

7.0 g/L K₂ HPO₄

2.0 g/L (NH₄)₂ SO₄

1.0 g/L Na₃ Citrate. 2H₂ O

153 mg/L MgSO₄, .7H₂ O

up to 2.30 g/L of L-proline,

2.5 mL/L of a trace metal solution containing:

32.5 μg/ml FeCl₃.6H₂ O

1.56 μg/ml ZnCl₂

2.4 μg/ml CoCl₂.6H₂ O, 2.4 μg/ml Na₂ MoO₄

2H₂ O,

1.22 mg/mL CaCl₂.2H₂ O,

1.54 μg/ml Cu(II)SO₄.5H₂ O,

0.6 μg/ml H₃ BO₃,

120 μl/ml HCl dissolved in purified water

After sterilization of the above solution, the following components were added to achieve the final concentrations indicated:

20 mL/L 10% yeast extract/L

4.0 mL 60% glucose solution/L

0.06 mg/L of sterile-filtered 125 mg/mL thiamine HCl dissolved in purified water

0.1 mg/L of tetracycline in an ˜50% ethanol solution

Fermentor (1 5 L Volume)

Two flasks (approximately 900 mls total volume) of fermentor inoculum was then asceptically transferred to a 15-liter BioLaffite fermentor containing approximately 6.5 liters of a solution containing:

1.83 g/L KH₂ PO₄

3.27 g/L K₂ HPO₄

1.83 g/L (NH₄)₂ SO₄

After sterilization of the above solution, the following components were added to achieve the final concentrations indicated:

1.36 g/L Trisodium Citrate

1.36 g/L MgSO₄.7H₂ O

2.87 g/L proline

3.05 g/L of the Trace Metal solution described above

0.1 mg/L tetracycline in 50% ethanol solution,

0.06 mg/L thiamine HCl in purified water, sterile filtered solution

200 g/L of 70% glucose

50+10 g/L of 30% NH₄ OH

2 ml PPG 2000

The fermentor was run at 30±1° C., pH 6.8, controlling dissolved oxygen at 20% and glucose between 0-6 g/L. At OD 30±2, induction was achieved by adding approximately 5.2 mL of 100 mM isopropyl thiogalactoside (IPTG) and 6 mL of 50 mg/mL hemin. At 3 hours post induction, 8.0 mL of 50 mg/mL hemin was added and at 6 hours post induction, 10 mL of 50 mg/mL hemin was added. Harvest and further purification occurs at 10 hours post induction. Other suitable fermentation conditions are described in Looker, D. et al., (1994) Meth. Enzymol. 231: 364-374.

C. Purification

Purification of expressed protein material was performed exactly as described by Looker, D. et al., (1994) Meth. Enzymol. 231: 364-374.

Example 2 Determination of Autooxidation Rates, Rate Constants and Ligand Affinities

Association and dissociation rates for O₂ and CO binding to the mutants were determined using conventional laser flash photolysis and stopped-flow rapid mixing techniques as described in detail by Rohlfs et al. (Rohlfs, R. J. et al., (1990) J. Biol. Chem. 265: 3168-3176). NO association rate constants were measured by photolyzing the NO-hemoglobin complexes at 25° C., pH 7.4 with an intense, 300 ns dye laser pulse at 578 nm (Phase-R 2100B) and following the time course for bimolecular recombination on appropriate time scales at 436 nm (Mathews, A. J. and Olson, J. S. (1994) Meth. Enzymol. 232: 363-386). NO dissociation reactions were carried out essentially as described by Moore and Gibson (Moore, E. G. and Gibson, Q. H. (1976) J. Biol. Chem. 251: 2788-2794). The mutant recombinant hemoglobin samples were prepared for determination of the NO dissociation rates by exposure of deoxygenated hemoglobin samples to NO followed by removal of excess NO by flushing the sample with nitrogen gas or by passage down a small Sephadex G-25 column equilibrated with CO-saturated sample buffer. NO dissociation was monitored in a Shimadzu UV-2101 spectrophotometer or a Hewlett-Packard HP8452 diode array spectrophotomer equipped with a temperature controlled multi-cell positioner. A concentrated, dithionite-free sample of NO-hemoglobin was injected into a sealed, 1 cm pathlength cuvette containing buffer, a bubble of pure CO, and a large excess of sodium dithionite to consume nitric oxide rapidly. The formation of the CO complex was monitored at 420-423 nm and, under these conditions, was limited by the rate of NO dissociation from the protein. The hemoglobin concentrations were 20-100 uM for the association experiments and 2-10 uM for the dissociation experiments. The buffer conditions were 0.1M potassium phosphate, pH 7.0, 20° C. or 50 mM HEPES, 100 mM NaCl, pH 7.4, 25° C.

Equilibrium association constants for O₂, CO, and NO binding to R-state hemoglobin were computed as the ratios of the overall rate constants. Autooxidation rates for hemoglobin were measured using the techniques described by Brantley et al. (Brantley, R. E., et al. (1993) J. Biol. Chem. 268: 6995-7010).

Example 3 Mutant Hemoglobins with Reduced Affinity for Nitric Oxide

Mutant recombinant hemoglobins containing Leu-B10→Phe mutations in either the alpha subunit or both the alpha and beta subunits were prepared as described in Example 1. Nitric oxide affinity was measured as described in Example 2. Only one NO association rate was observed for the photolysis of αLeu29Phe containing hemoglobins, and this corresponded to the rate observed for isolated αLeu29Phe subunits. This result suggests a large increase in the apparent quantum yield for these subunits relative to rHb0.0 and βLeu28Phe subunits. Consequently, beta subunit rate constants could not be obtained for any hemoglobin containing αLeu29Phe subunits.

The Leu B10Phe mutation caused a large 9.5-fold decrease in the alpha subunit NO association rate, but only 1.5-fold decrease in the beta subunit NO association rate, which is not a significant change in light of variation of the data (Table 2). The larger change in the alpha subunit rate due to the Leu B10Phe mutation is a reflection of the intrinsically more sterically hindered nature of the alpha subunit distal heme pocket relative to that in beta subunits. The Leu B10 Phe mutation in alpha subunits caused only a small 2.6-fold increase in the rate of NO dissociation relative to rHb0.0 alpha subunits. The rate of NO dissociation from 0.0 beta subunits was not determined, but relative to human beta subunits, the rate of NO dissociation from βLeu28Phe subunits was increased 11.1-fold (using the average of two determinations for βLeu28Phe subunits). In terms of the association equilibrium constants, the LeuB10Phe mutation causes a 14-fold decrease in alpha subunit NO affinity, and a 4-fold decrease in beta subunit NO affinity with respect to the affinities observed for R-state native hemoglobin (Table 3). The net consequence of these mutations in hemoglobin [(α0.0)(βLeu28Phe)]₂ is a significant decrease in NO affinity relative to that of human hemoglobin A.

A protocol similar to Example 1 was followed to prepare a mutant recombinant beta subunit except that the mutant codon coded for Trp rather than the wild type Leu. Nitric oxide affinity in these mutant beta subunits was decreased further when Leu-B1O was replaced with the even large Trp residue (Table 2). An even larger decrease in reactivity occurs for the same Trp mutation in alpha subunits. A combination of Ile-E11 and Phe or Trp-B10 mutations markedly inhibits the binding of all three gases by eliminating any available space for bound ligands.

Example 4 Mutant Hemoglobins with Enhanced Affinity for Nitric Oxide

Mutant recombinant hemoglobins containing His-E7→Leu or His-E7→Phe mutations in either the alpha globin, the beta globin or both the alpha and beta globin are constructed as described in Example 1. Nitric oxide affinity is measured as described in Example 2. Replacing His-E7 with Leu or Phe causes large increases in NO affinity because water is already displaced from the distal pocket in the deoxy alpha and beta subunits. The Val-E11 to Leu mutation sterically displaces distal pocket water molecules and can also enhance NO binding. Combinations of these mutations, particularly the ValE11→Phe/His-E7→Leu double mutant produce hemoglobin subunits and hemoglobin tetramers with even higher NO affinities.

Example 5 Mutant Hemoglobins with Reduced Affinity for Carbon Monoxide

Mutant recombinant hemoglobins containing Leu-B10→Phe mutations in either the alpha subunit or both the alpha and beta subunits were prepared as described in Example 1. Carbon monoxide affinity was measured as described in Example 2. The Leu-B10 to Phe mutation caused marked decreases in CO affinity 40- and 10-fold in the alpha and beta subunits, respectively (Table 3). Similar reductions in CO affinity were observed for the Leu-B10 to Trp mutation. Curiously, the Leu-B10 to Ile mutation had no effect in alpha subunits but decreased the affinity of beta subunits ˜10-fold (Table 3). Mutations at the CD3 site in beta globin had little effect on CO affinity. A variety of other Val-E11 substitutions reduce CO affinity including Phe-E11, Asn-E11, Gln-E11, and Trp-E11. Replacement of Phe-CD1 in beta subunits and Phe-CE1 in alpha subunits, particularly with Trp reduce CO affinity significantly. The largest decreases in CO affinity occur for Phe-B10/Ile-E11 and Phe-B10/Asn-E11 double mutants.

Example 6 Mutant Hemoglobins with Enhanced Affinity for Carbon Monoxide

Mutant recombinant hemoglobins containing His-E7→Leu or Phe and/or Val-E11→Leu in either the alpha globin, the beta globin or both the alpha and beta globin are prepared as described in Example 1. Carbon monoxide affinity is measured as described in Example 2. The affinity of hemoglobin subunits for CO is enhanced by excluding distal pocket water without enhancing steric hindrance in the vicinity of the iron atom. Large increases occur when His-E7 is replaced with Leu or Phe, particularly in alpha subunits. The largest increases occur when the E7 and E11 residues are both leucines.

Example 7 Mutant Hemoglobins with Reduced Autooxidation and Oxidation by NO

Mutant recombinant hemoglobins containing Leu-B 10→Phe mutations in either the alpha subunit or both the alpha and beta subunits are prepared as described in Example 1. Mutant recombinant hemoglobins containing the mutations described below are prepared in a fashion analogous to Example 1, and autooxidation rate measurements are performed as described in Example 2. Autooxidation and NO induced oxidation rates are reduced by excluding solvents or other oxidizing or protonating agents from the distal pocket and increasing the apolar character of the distal pocket (FIG. 1). This is achieved by placing large aromatic residues at the B10, E11, and the B13, G8, and G12 positions in the back of the distal pocket. Phe-B10 or Phe-E11 substitutions in both hemoglobin subunits maintain low rates of oxidation while changing oxygen affinity (FIG. 2A). The Phe-B10/Phe-E11 and Phe-B10/Leu-E11 combinations reduce oxidation rates even further. His E7→Gln weakens the hydrogen bond to the bound oxygen, lowering oxygen affinity but at the same time increasing the rate of oxidation (FIG. 2B). The His E7-→Gln/Leu B10→Phe double mutant shows optimum oxygen binding with low autooxidation and oxidation by NO (FIG. 2C).

                  TABLE 1                                                          ______________________________________                                         AMINO ACID SEQUENCE AND HELICAL RESIDUE                                        NOTATION FOR HUMAN HEMOGLOBIN Ao                                               Helix   α        Helix  β                                           ______________________________________                                         NA1     1Val           NA1    1 Val                                                                   NA2    2 His                                            NA2     2 Leu          NA3    3 Leu                                            A1      3 Ser          A1     4 Thr                                            A2      4 Pro          A2     5 Pro                                            A3      5 Ala          A3     6 Glu                                            A4      6 Asp          A4     7 Glu                                            A5      7 Lys          A5     8 Lys                                            A6      8 Thr          A6     9 Ser                                            A7      9 Asn          A7     10 Ala                                           A8      10 Val         A8     11 Val                                           A9      11 Lys         A9     12 Thr                                           A10     12 Ala         A10    13 Ala                                           A11     13 Ala         A11    14 Leu                                           A12     14 Trp         A12    15 Trp                                           A13     15 Gly         A13    16 Gly                                           A14     16 Lys         A14    17 Lys                                           A15     17 Val         A15    18 Val                                           A16     18 Gly                                                                 AB1     19 Ala                                                                 B1      20 His         B1     19 Asn                                           B2      21 Ala         B2     20 Val                                           B3      22 Gly         B3     21 Asp                                           B4      23 Glu         B4     22 Glu                                           B5      24 Tyr         B5     23 Val                                           B6      25 Gly         B6     24 Gly                                           B7      26 Ala         B7     25 Gly                                           B8      27 Glu         B8     26 Glu                                           B9      28 Ala         B9     27 Ala                                           B10     29 Leu         B10    28 Leu                                           B11     30 Glu         B11    29 Gly                                           B12     31 Arg         B12    30 Arg                                           B13     32 Met         B13    31 Leu                                           B14     33 Phe         B14    32 Leu                                           B15     34 Leu         B15    33 Val                                           B16     35 Ser         B16    34 Val                                           C1      36 Phe         C1     35 Tyr                                           C2      37 Pro         C2     36 Pro                                           C3      38 Thr         C3     37 Trp                                           C4      39 Thr         C4     38 Thr                                           C5      40 Lys         C5     39 Gln                                           C6      41 Thr         C6     40 Arg                                           C7      42 Tyr         C7     41 Phe                                           CE1     43 Phe         CD1    42 Phe                                           CE2     44 Pro         CD2    43 Glu                                           CE3     45 His         CD3    44 Ser                                           CE4     46 Phe         CD4    45 Phe                                                                  CD5    46 Gly                                           CE5     47 Asp         CD6    47 Asp                                           CE6     48 Leu         CD7    48 Leu                                           CE7     49 Ser         CD8    49 Ser                                           CE8     50 His         D1     50 Thr                                                                  D2     51 Pro                                                                  D3     52 Asp                                                                  D4     53 Ala                                                                  D5     54 Val                                                                  D6     55 Met                                           CE9     51 Gly         D7     56 Gly                                           E1      52 Ser         E1     57 Asn                                           E2      53 Ala         E2     58 Pro                                           E3      54 Gln         E3     59 Lys                                           E4      55 Val         E4     60 Val                                           E5      56 Lys         E5     61 Lys                                           E6      57 Gly         E6     62 Ala                                           E7      58 His         E7     63 His                                           E8      59 Gly         E8     64 Gly                                           E9      60 Lys         E9     65 Lys                                           E10     61 Lys         E10    66 Lys                                           E11     62 Val         E11    67 Val                                           E12     63 Ala         E12    68 Leu                                           E13     64 Asp         E13    69 Gly                                           E14     65 Ala         E14    70 Ala                                           E15     66 Leu         E15    71 Phe                                           E16     67 Thr         E16    72 Ser                                           E17     68 Asn         E17    73 Asp                                           E18     69 Ala         E18    74 Gly                                           E19     70 Val         E19    75 Leu                                           E20     71 Ala         E20    76 Ala                                           EF1     72 His         EF1    77 His                                           EF2     73 Val         EF2    78 Leu                                           EF3     74 Asp         EF3    79 Asp                                           EF4     75 Asp         EF4    80 Asn                                           EF5     76 Met         EF5    81 Leu                                           EF6     77 Pro         EF6    82 Lys                                           EF7     78 Asn         EF7    83 Gly                                           EF8     79 Ala         EF8    84 Thr                                           F1      80 Leu         F1     85 Phe                                           F2      81 Ser         F2     86 Ala                                           F3      82 Ala         F3     87 Thr                                           F4      83 Leu         F4     88 Leu                                           F5      84 Ser         F5     89 Ser                                           F6      85 Asp         F6     90 Glu                                           F7      86 Leu         F7     91 Leu                                           F8      87 His         F8     92 His                                           F9      88 Ala         F9     93 Cys                                           FG1     89 His         FG1    94 Asp                                           FG2     90 Lys         FG2    95 Lys                                           FG3     91 Leu         FG3    96 Leu                                           FG4     92 Arg         FG4    97 His                                           FG5     93 Val         FG5    98 Val                                           G1      94 Asp         G1     99Asp                                            G2      95 Pro         G2     100 Pro                                          G3      96 Val         G3     101 Glu                                          G4      97 Asn         G4     102 Asn                                          G5      98 Phe         G5     103 Phe                                          G6      99 Lys         G6     104 Arg                                          G7      100 Leu        G7     105 Leu                                          G8      101 Leu        G8     106 Leu                                          G9      102 Ser        G9     107 Gly                                          G10     103 His        G10    108 Asn                                          G11     104 Cys        G11    109 Val                                          G12     105 Leu        G12    110 Leu                                          G13     106 Leu        G13    111 Val                                          G14     107 Val        G14    112 Cys                                          G15     108 Thr        G15    113 Val                                          G16     109 Leu        G16    114 Leu                                          G17     110 Ala        G17    115 Ala                                          G18     111 Ala        G18    116 His                                          G19     112 His        G19    117 His                                          GH1     113 Leu        GH1    118 Phe                                          GH2     114 Pro        GH2    119 Gly                                          GH3     115 Ala        GH3    120 Lys                                          GH4     116 Glu        GH4    121 Glu                                          GH5     117 Phe        GH5    122 Phe                                          H1      118 Thr        H1     123 Thr                                          H2      119 Pro        H2     124 Pro                                          H3      120 Ala        H3     125 Pro                                          H4      121 Val        H4     126 Val                                          H5      122 His        H5     127 Gln                                          H6      123 Ala        H6     128 Ala                                          H7      124 Ser        H7     129 Ala                                          H8      125 Leu        H8     130 Tyr                                          H9      126 Asp        H9     131 Gln                                          H10     127 Lys        H10    132 Lys                                          H11     128 Phe        H11    133 Val                                          H12     129 Leu        H12    134 Val                                          H13     130 Ala        H13    135 Ala                                          H14     131 Ser        H14    136 Gly                                          H15     132 Val        H15    137 Val                                          H16     133 Ser        H16    138 Ala                                          H17     134 Thr        H17    139 Asn                                          H18     135 Val        H18    140 Ala                                          H19     136 Leu        H19    141 Leu                                          H20     137 Thr        H20    142 Ala                                          H21     138 Ser        H21    143 His                                          HC1     139 Lys        HC1    144 Lys                                          HC2     140 Tyr        HC2    145 Tyr                                          HC3     141 Arg        HC3    146 His                                          ______________________________________                                    

                  TABLE 2                                                          ______________________________________                                         Effects of distal pocket mutations on NO binding to isolated α and       β                                                                         subunits and α and β                                                subunits within R-state recombinant hemoglobins at pH 7.4, 25° C.                 k'.sub.NO                                                                             k.sub.NO      K.sub.NO                                                  μM.sup.-1 s.sup.-1                                                                 s.sup.-1      nM.sup.-1                                       Protein     α                                                                              β α                                                                               β α                                                                             β                             ______________________________________                                         Native hemoglobin A                                                                        16    34     0.000028                                                                              0.000058                                                                              570  590                                Native isolated α                                                                    29                                                                 subunits                                                                       Native isolated β                                                                           110                                                          subunits                                                                       rHb 0.0     36    140                                                          α 0.0 isolated                                                                       34                                                                 β 0.0 isolated                                                                              120                                                          [(αLeu 29 -> Phe/                                                                    3.6   N/A                                                          0.0 beta subunit)].sub.2                                                       [(0.0 alpha subunit/                                                                       36    95     0.000037                                                                              0.00058                                                                               970  160                                βLeu 28 -> Phe)].sub.2                                                    [(αLeu-29 -> Phe/                                                                    3.9   N/A    0.000098                                                                              0.00071                                                                               40   130                                βLeu 28 -> Phe)].sub.2                                                    αLeu 29 -> Phe,                                                                      5.5                                                                isolated                                                                       βLeu 28 ->Phe,                                                                              83                                                           isolated                                                                       ______________________________________                                    

Note that the designation, for example, [(αLeu -29→Phe/βLeu 28→Phe)]₂ indicates a tetrameric hemoglobin composed of 2 alpha/beta dimers containing the indicated mutations. This notation is used throughout the table.

                  TABLE 3                                                          ______________________________________                                         Effects of distal pocket mutations on O.sub.2 and CO binding to α        and β                                                                     subunits within R-state recombinant hemoglobins at pH 7, 20° C.                       k'.sub.O2 k.sub.O2                                                                               K.sub.O2                                                                              K.sub.CO                                Protein       μM.sup.-1 s.sup.-1                                                                    s.sup.-1                                                                               μM.sup.-1                                                                          μM.sup.-1                            ______________________________________                                         α (0.0) 19        15      1.3    300                                     α (Leu B10 -> Ile)                                                                     6.0       6.9     0.9    200                                     α (Leu B10 -> Phe)                                                                     0.9.sup.a 0.3.sup.a                                                                              3.sup.a                                                                               7.sup.a                                 β (0.0)  74        47      1.6    400                                     β (Leu B10 -> Ile)                                                                      27        4.6     6      40                                      β (Leu B10 -> Phe)                                                                      8.sup.a   12.sup.a                                                                               0.7.sup.a                                                                             30.sup.a                                β (Leu B10 -> Trp)                                                                      0.016     0.50    0.03   40                                      β (Ser CD3 -> His)                                                                      92        24      4      500                                     β (Ser CD3 -> Lys)                                                                      76        29      3      300                                     β (Ser CD3 -> Arg)                                                                      87        30      3      300                                     ______________________________________                                          k'.sub.O2 = is the association rate constant for the reaction of oxygen        with Rstate hemoglobin                                                         k.sub.O2 = is the dissociation rate constant for the dissociation of           oxygen from Rstate hemoglobin                                                  K.sub.O2 = is the association equilibrium constant (affinity) for the          binding of oxygen to Rstate hemoglobin, calculated as the ratio of             k'.sub.O2 to k.sub.O2.                                                         K.sub.CO = is the association equilibrium constant (affinity) for the          binding of carbon monoxide to Rstate hemoglobin.                               0.0 refers to hemoglobin expressed in the pSGE0.0E4 plasmid and is             described above.                                                               .sup.a The conditions for the PheB10 mutant experiments were pH 7.4,           25° C.                                                            

    __________________________________________________________________________     #             SEQUENCE LISTING                                                 - (1) GENERAL INFORMATION:                                                     -    (iii) NUMBER OF SEQUENCES:  8                                             - (2) INFORMATION FOR SEQ ID NO:1:                                             -      (i) SEQUENCE CHARACTERISTICS:                                                     (A) LENGTH:  141                                                               (B) TYPE:  amino aci - #d                                                      (D) TOPOLOGY:  linear                                                -     (ii) MOLECULE TYPE:  protein                                             -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #1:                             - Val Leu Ser Pro Ala Asp Lys Thr Asn Val Ly - #s Ala Ala Trp Gly              #15                                                                            - Lys Val Gly Ala His Ala Gly Glu Tyr Gly Al - #a Glu Ala Leu Glu              #                305                                                           - Arg Met Phe Leu Ser Phe Pro Thr Thr Lys Th - #r Tyr Phe Pro His              #                 45                                                           - Phe Asp Leu Ser His Gly Ser Ala Gln Val Ly - #s Gly His Gly Lys              #                 60                                                           - Lys Val Ala Asp Ala Leu Thr Asn Ala Val Al - #a His Val Asp Asp              #                 75                                                           - Met Pro Asn Ala Leu Ser Ala Leu Ser Asp Le - #u His Ala His Lys              #                 90                                                           - Leu Arg Val Asp Pro Val Asn Phe Lys Leu Le - #u Ser His Cys Leu              #                105                                                           - Leu Val Thr Leu Ala Ala His Leu Pro Ala Gl - #u Phe Thr Pro Ala              #               120                                                            - Val His Ala Ser Leu Asp Lys Phe Leu Ala Se - #r Val Ser Thr Val              #               135                                                            - Leu Thr Ser Lys Tyr Arg                                                                      140                                                            - (2) INFORMATION FOR SEQ ID NO:2:                                             -      (i) SEQUENCE CHARACTERISTICS:                                                     (A) LENGTH:  146                                                               (B) TYPE:  amino aci - #d                                                      (D) TOPOLOGY: unknown t - #o applicant                               -     (ii) MOLECULE TYPE:  protein                                             -    (iii) HYPOTHETICAL:  no                                                   -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #2:                             - Val His Leu Thr Pro Glu Glu Lys Ser Ala Va - #l Thr Ala Leu Trp              #15                                                                            - Gly Lys Val Asn Val Asp Glu Val Gly Gly Gl - #u Ala Leu Gly Arg              #                 30                                                           - Leu Leu Val Val Tyr Pro Trp Thr Gln Arg Ph - #e Phe Glu Ser Phe              #                 45                                                           - Gly Asp Leu Ser Thr Pro Asp Ala Val Met Gl - #y Asn Pro Lys Val              #                 60                                                           - Lys Ala His Gly Lys Lys Val Leu Gly Ala Ph - #e Ser Asp Gly Leu              #                 75                                                           - Ala His Leu Asp Asn Leu Lys Gly Thr Phe Al - #a Thr Leu Ser Glu              #                 90                                                           - Leu His Cys Asp Lys Leu His Val Asp Pro Gl - #u Asn Phe Arg Leu              #                105                                                           - Leu Gly Asn Val Leu Val Cys Val Leu Ala Hi - #s His Phe Gly Lys              #               120                                                            - Glu Phe Thr Pro Pro Val Gln Ala Ala Tyr Gl - #n Lys Val Val Ala              #               135                                                            - Gly Val Ala Asn Ala Leu Ala His Lys Tyr Hi - #s                              #               145                                                            - (2) INFORMATION FOR SEQ ID NO:3:                                             -      (i) SEQUENCE CHARACTERISTICS:                                                     (A) LENGTH:  423                                                               (B) TYPE:  nucleic a - #cid                                                    (C) STRANDEDNESS:  sing - #le                                                  (D) TOPOLOGY: unknown t - #o applicant                               -     (ii) MOLECULE TYPE:  Other nucleic aci - #d                                        (A) DESCRIPTION:  Human - # alpha globin sequence                    -    (iii) HYPOTHETICAL:  no                                                   -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #3:                             #              50CGACAA GACCAACGTC AAGGCCGCCT GGGGCAAGGT                       #             100GCGAGT ATGGTGCGGA GGCCCTGGAG AGGATGTTCC                       #             150ACCAAG ACCTACTTCC CGCACTTCGA CCTGAGCCAC                       #             200TAAGGG CCACGGCAAG AAGGTGGCCG ACGCGCTGAC                       #             250ACGTGG ACGACATGCC CAACGCGCTG TCCGCCCTGA                       #             300CACAAG CTTCGGGTGG ACCCGGTCAA CTTCAAGCTC                       #             350GCTGGT GACCCTGGCC GCCCACCTCC CCGCCGAGTT                       #             400ACGCCT CCCTGGACAA GTTCCTGGCT TCTGTGAGCA                       #               423ATAC CGT                                                    - (2) INFORMATION FOR SEQ ID NO:4:                                             -      (i) SEQUENCE CHARACTERISTICS:                                                     (A) LENGTH:  438                                                               (B) TYPE:  nucleic a - #cid                                                    (C) STRANDEDNESS:  sing - #le                                                  (D) TOPOLOGY: unknown t - #o applicant                               -     (ii) MOLECULE TYPE:  Other nucleic aci - #d                                        (A) DESCRIPTION:  Human - # beta globin sequence                     -    (iii) HYPOTHETICAL:  no                                                   -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #4:                             #              50TGAGGA GAAGTCTGCC GTTACTGCCC TGTGGGGCAA                       #             100AAGTTG GTGGTGAGGC CCTGGGCAGG CTGCTGGTGG                       #             150CAGAGG TTCTTTGAGT CCTTTGGGGA TCTGTCCACT                       #             200GGGCAA CCCTAAGGTG AAGGCTCATG GCAAGAAAGT                       #             250GTGATG GCCTGGCTCA CCTGGACAAC CTCAAGGGCA                       #             300AGTGAG CTGCACTGTG ACAAGCTGCA CGTGGATCCT                       #             350CCTGGG CAACGTGCTG GTCTGTGTGC TGGCCCATCA                       #             400TCACCC CACCAGTGCA GGCTGCCTAT CAGAAAGTGG                       #    438           TGCC CTGGCCCACA AGTATCAC                                    - (2) INFORMATION FOR SEQ ID NO:5:                                             -      (i) SEQUENCE CHARACTERISTICS:                                                     (A) LENGTH:  423                                                               (B) TYPE:  nucleic a - #cid                                                    (C) STRANDEDNESS:  sing - #le                                                  (D) TOPOLOGY: unknown t - #o applicant                               -     (ii) MOLECULE TYPE:  Other nucleic aci - #d                                        (A) DESCRIPTION:  Human - # alpha globin sequence for expression     #E. co           in                                                            -    (iii) HYPOTHETICAL:  no                                                   -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #5:                             #              50CGATAA AACCAACGTT AAAGCTGCTT GGGGTAAAGT                       #             100GTGAAT ACGGTGCTGA AGCTCTCGAG CGTATGTTCC                       #             150ACCAAA ACCTACTTCC CGCACTTCGA CCTGTCTCAC                       #             200TAAAGG TCACGGTAAA AAAGTTGCTG ATGCTCTGAC                       #             250ACGTTG ATGATATGCC GAACGCGTTG TCTGCTCTGT                       #             300CACAAA CTGCGTGTTG ATCCGGTTAA CTTCAAACTG                       #             350GCTGGT TACTCTGGCT GCTCATCTGC CGGCTGAATT                       #             400ATGCGT CTCTGGATAA ATTCCTGGCT TCTGTTTCTA                       #               423ATAC CGT                                                    - (2) INFORMATION FOR SEQ ID NO:6:                                             -      (i) SEQUENCE CHARACTERISTICS:                                                     (A) LENGTH:  438                                                               (B) TYPE:  nucleic a - #cid                                                    (C) STRANDEDNESS:  sing - #le                                                  (D) TOPOLOGY: unknown t - #o applicant                               -     (ii) MOLECULE TYPE:  Other nucleic aci - #d                                        (A) DESCRIPTION:  Human - # beta globin sequence for expression      #E. col          in                                                            -    (iii) HYPOTHETICAL:  no                                                   -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #6:                             #              50GGAAGA AAAATCCGCG GTTACTGCTC TGTGGGGTAA                       #             100AAGTTG GTGGTGAAGC TCTGGGACGT CTGCTGGTTG                       #             150CAGCGT TTCTTTGAAT CTTTCGGAGA TCTGTCTACC                       #             200GGGTAA CCCGAAAGTT AAAGCCCATG GTAAAAAAGT                       #             250CTGACG GTCTGGCTCA CCTGGACAAC CTGAAAGGTA                       #             300TCTGAG CTCCACTGCG ACAAACTGCA CGTTGACCCG                       #             350GCTGGG TAAAGTACTA GTTTGCGTTC TGGCTCACCA                       #             400TCACTC CGCCGGTTCA GGCTGCTTAC CAGAAAGTTG                       #    438           CGCG CTAGCTCACA AATACCAC                                    - (2) INFORMATION FOR SEQ ID NO:7:                                             -      (i) SEQUENCE CHARACTERISTICS:                                                     (A) LENGTH:  141                                                               (B) TYPE:  amino aci - #d                                                      (D) TOPOLOGY: unknown t - #o applicant                               -     (ii) MOLECULE TYPE:                                                                (A) DESCRIPTION:  alpha - # globin protein sequence as expressed     #E. co           in                                                            -    (iii) HYPOTHETICAL:  no                                                   -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #7:                             - Met Leu Ser Pro Ala Asp Lys Thr Asn Val Ly - #s Ala Ala Trp Gly              #15                                                                            - Lys Val Gly Ala His Ala Gly Glu Tyr Gly Al - #a Glu Ala Leu Glu              #                 30                                                           - Arg Met Phe Leu Ser Phe Pro Thr Thr Lys Th - #r Tyr Phe Pro His              #                 45                                                           - Phe Asp Leu Ser His Gly Ser Ala Gln Val Ly - #s Gly His Gly Lys              #                 60                                                           - Lys Val Ala Asp Ala Leu Thr Asn Ala Val Al - #a His Val Asp Asp              #                 75                                                           - Met Pro Asn Ala Leu Ser Ala Leu Ser Asp Le - #u His Ala His Lys              #                 90                                                           - Leu Arg Val Asp Pro Val Asn Phe Lys Leu Le - #u Ser His Cys Leu              #                105                                                           - Leu Val Thr Leu Ala Ala His Leu Pro Ala Gl - #u Phe Thr Pro Ala              #               120                                                            - His Val Ala Ser Leu Asp Lys Phe Leu Ala Se - #r Val Ser Thr Val              #               135                                                            - Leu Thr Ser Lys Tyr Arg                                                                      140                                                            - (2) INFORMATION FOR SEQ ID NO:8:                                             -      (i) SEQUENCE CHARACTERISTICS:                                                     (A) LENGTH:  146                                                               (B) TYPE:  amino aci - #d                                                      (D) TOPOLOGY: unknown t - #o applicant                               -     (ii) MOLECULE TYPE:                                                      #globin protein sequence as expressed                                          #E. col          in                                                            -    (iii) HYPOTHETICAL:  no                                                   -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #8:                             - Met His Leu Thr Pro Glu Glu Lys Ser Ala Va - #l Thr Ala Leu Trp              #15                                                                            - Gly Lys Val Asn Val Asp Glu Val Gly Gly Gl - #u Ala Leu Gly Arg              #                 30                                                           - Leu Leu Val Val Tyr Pro Trp Thr Gln Arg Ph - #e Phe Glu Ser Phe              #                 45                                                           - Gly Asp Leu Ser Thr Pro Asp Ala Val Met Gl - #y Asn Pro Lys Val              #                 60                                                           - Lys Ala His Gly Lys Lys Val Leu Gly Ala Ph - #e Ser Asp Gly Leu              #                 75                                                           - Ala His Leu Asp Asn Leu Lys Gly Thr Phe Al - #a Thr Leu Ser Glu              #                 90                                                           - Leu His Cys Asp Lys Leu His Val Asp Pro Gl - #u Asn Phe Arg Leu              #                105                                                           - Leu Gly Lys Val Leu Val Cys Val Leu Ala Hi - #s His Phe Gly Lys              #               120                                                            - Glu Phe Thr Pro Pro Val Gln Ala Ala Tyr Gl - #n Lys Val Val Ala              #               135                                                            - Gly Val Ala Asn Ala Leu Ala His Lys Tyr Hi - #s                              #               145                                                            __________________________________________________________________________ 

We claim:
 1. A mutant recombinant hemoglobin comprising a recombinant hemoglobin with one or more mutations of any amino acid of a heme pocket of said recombinant hemoglobin wherein said mutation is not B10 (Leu→Gln) or CD3 (Ser→Arg) of the β chain.
 2. A mutant recombinant hemoglobin according to claim 1 wherein said mutations occur within 4 Å of a heme moiety bound within said heme pocket.
 3. A mutant recombinant hemoglobin according to claim 1 wherein said mutations occur in that portion of said heme pocket that is a distal heme pocket.
 4. A mutant recombinant hemoglobin according to claim 1 wherein said mutant recombinant hemoglobin has lower autooxidation rates than autooxidation rates measured for naturally occurring purified human hemoglobin under comparable conditions.
 5. A mutant recombinant hemoglobin according to claim 1 wherein said mutant recombinant hemoglobin has either higher nitric oxide affinity than nitric oxide affinity measured for naturally occurring purified human hemoglobin under comparable conditions or has a lower rate of oxidation by NO than NO oxidation rate measured for naturally occurring purified human hemoglobin under comparable conditions.
 6. A mutant recombinant hemoglobin according to claim 1 wherein said mutant recombinant hemoglobin has lower nitric oxide affinity than nitric oxide affinity measured for naturally occurring purified human hemoglobin under comparable conditions.
 7. A mutant recombinant hemoglobin according to claim 1 wherein said mutant recombinant hemoglobin has lower carbon monoxide affinity than carbon monoxide affinity measured for naturally occurring purified human hemoglobin under comparable conditions.
 8. A mutant recombinant hemoglobin according to claim 1 wherein said mutant recombinant hemoglobin has higher carbon monoxide affinity than carbon monoxide affinity measured for naturally occurring purified human hemoglobin under comparable conditions.
 9. A mutant recombinant hemoglobin according to claim 4 wherein said one or more amino acid is selected from the group consisting of B10 Leu, B113 Met, CE1 Phe, CE3 His, E7 His, CE4 Phe, E11 Val, E10 Lys, G8 Leu, E14 Ala, G12 Leu, F8 His, C7 Tyr, F4 Leu, F7 Leu, FG3 Leu, FG5 Val, G4 Asn, G5 Phe, H15 Val and H19 Leu for alpha globin and B10 Leu, B13 Leu, CD1 Phe, CD3 Ser, E7 His, CD4 Phe, E11 Val, E10 Lys, G8 Leu, E14 Ala, G12 Leu, F8 His, C7 Phe, F4 Leu, F7 Leu, FG3 Leu, FG5 Val, G4 Asn, G5 Phe, H15 Val and H19 Leu for beta globin.
 10. A mutant recombinant hemoglobin according to claim 5 wherein said one or more amino acid is selected from the group consisting of B10 Leu, B13 Met, CE1 Phe, CE3 His, E7 His, CE4 Phe, E11 Val, E10 Lys, G8 Leu, E14 Ala, G12 Leu, F8 His, C7 Tyr, F4 Leu, F7 Leu, FG3 Leu, FG5 Val, G4 Asn, G5 Phe, H15 Val and H19 Leu for alpha globin and B10 Leu, B13 Leu, CD1 Phe, CD3 Ser, E7 His, CD4 Phe, E11 Val, E10 Lys, G8 Leu, E14 Ala, G12 Leu, F8 His, C7 Phe, F4 Leu, F7 Leu, FG3 Leu, FG5 Val, G4 Asn, G5 Phe, H15 Val and H19 Leu for beta globin.
 11. A mutant recombinant hemoglobin according to claim 6 wherein said one or more amino acid is selected from the group consisting of B10 Leu, B13 Met, CE1 Phe, CE3 His, E7 His, CE4 Phe, E11 Val, E10 Lys, G8 Leu, E14 Ala, G12 Leu, F8 His, C7 Tyr, F4 Leu, F7 Leu, FG3 Leu, FG5 Val, G4 Asn, G5 Phe, H15 Val and H19 Leu for alpha globin and B10 Leu, B13 Leu, CD1 Phe, CD3 Ser, E7 His, CD4 Phe, E11 Val, E10 Lys, G8 Leu, E14 Ala, G12 Leu, F8 His, C7 Phe, F4 Leu, F7 Leu, FG3 Leu, FG5 Val, G4 Asn, G5 Phe, H15 Val and H19 Leu for beta globin.
 12. A mutant recombinant hemoglobin according to claim 7 wherein said one or more amino acid is selected from the group consisting of B10 Leu, B13 Met, CE1 Phe, CE3 His, E7 His, CE4 Phe, E11 Val, E10 Lys, G8 Leu, E14 Ala, G12 Leu, F8 His, C7 Tyr, F4 Leu, F7 Leu, FG3 Leu, FG5 Val, G4 Asn, G5 Phe, H15 Val and H19 Leu for alpha globin and B10 Leu, B13 Leu, CD1 Phe, CD3 Ser, E7 His, CD4 Phe, E11 Val, E10 Lys, G8 Leu, E14 Ala, G12 Leu, F8 His, C7 Phe, F4 Leu, F7 Leu, FG3 Leu, FG5 Val, G4 Asn, G5 Phe, H15 Val and H19 Leu for beta globin.
 13. A mutant recombinant hemoglobin according to claim 8 wherein said one or more amino acid is selected from the group consisting of B10 Leu, B13 Met, CE1 Phe, CE3 His, E7 His, CE4 Phe, E11 Val, E10 Lys, G8 Leu, E14 Ala, G12 Leu, F8 His, C7 Tyr, F4 Leu, F7 Leu, FG3 FG5 Val, G4 Asn, G5 Phe, H15 Val and H19 Leu for alpha globin and B10 Leu, B13 Leu, CD1 Phe, CD3 Ser, E7 His, CD4 Phe, E11 Val, E10 Lys, G8 Leu, E14 Ala, G12 Leu, F8 His, C7 Phe, F4 Leu, F7 Leu, FG3 Leu, FG5 Val, G4 Asn, G5 Phe, H15 Val and H19 Leu for beta globin.
 14. A mutant recombinant hemoglobin according to claim 9 wherein said one or more amino acid is selected from the group consisting of B10 Leu, B13 Met, CE1 Phe, CE3 His, E7 His, CE4 Phe, E11 Val, E10 Lys, G8 Leu, E14 Ala, and G12 Leu for alpha globin and B10 Leu, B13 Leu, CD1 Phe, CD3 Ser, E7 His, CD4 Phe, E11 Val, E10 Lys, G8 Leu, E14 Ala, and G12 Leu for beta globin.
 15. A mutant recombinant hemoglobin according to claim 10 wherein said one or more amino acid is selected from the group consisting of B10 Leu, B13 Met, CE1 Phe, CE3 His, E7 His, CE4 Phe, E11 Val, E10 Lys, G8 Leu, E14 Ala, and G12 Leu for alpha globin and B10 Leu, B13 Leu, CD1 Phe, CD3 Ser, E7 His, CD4 Phe, E11 Val, E10 Lys, G8 Leu, E14 Ala, and G12 Leu for beta globin.
 16. A mutant recombinant hemoglobin according to claim 11 wherein said one or more amino acid is selected from the group consisting of B10 Leu, B13 Met, CE1 Phe, CE3 His, E7 His, CE4 Phe, E11 Val, E10 Lys, G8 Leu, E14 Ala, and G12 Leu for alpha globin and B10 Leu, B13 Leu, CD1 Phe, CD3 Ser, E7 His, CD4 Phe, E11 Val, E10 Lys, G8 Leu, E14 Ala, and G12 Leu for beta globin.
 17. A mutant recombinant hemoglobin according to claim 12 wherein said one or more amino acid is selected from the group consisting of B10 Leu, B13 Met, CE1 Phe, CE3 His, E7 His, CE4 Phe, E11 Val, E 10 Lys, G8 Leu, E14 Ala, and G 12 Leu for alpha globin and B10 Leu, B 13 Leu, CD1 Phe, CD3 Ser, E7 His, CD4 Phe, E11 Val, E10 Lys, G8 Leu, E14 Ala, and G12 Leu for beta globin.
 18. A mutant recombinant hemoglobin according to claim 13 wherein said one or more amino acid is selected from the group consisting of B10 Leu, B13 Met, CE1 Phe, CE3 His, E7 His, CE4 Phe, E11 Val, E10 Lys, G8 Leu, E14 Ala, and G12 Leu for alpha globin and B10 Leu, B13 Leu, CD1 Phe, CD3 Ser, E7 His, CD4 Phe, E11 Val, E10 Lys, G8 Leu, E14 Ala, and G12 Leu for beta globin.
 19. A mutant recombinant hemoglobin according to claim 4 wherein one or more of the mutations are selected from the group consisting of Leu-B10→Phe, Val-E11→Phe and His-E7→Gln for alpha and/or beta globin.
 20. A mutant recombinant hemoglobin according to claim 5 wherein one or more of the mutations are selected from the group consisting of Leu-B10→Trp, Leu-B10→Phe, Leu-B10→Val, Leu-B10→Ile, Leu-B10→Ala, His-E7→Leu, His-E7→Trp, Val-E11→Phe, and Val E11→Trp for alpha and/or beta globin.
 21. A mutant recombinant hemoglobin according to claim 6 wherein one or more of the mutations are selected from the group consisting of Leu-B10→Trp, Leu-B10→Phe, Leu-B10→Val, Leu-B10→Ile, Leu-B10→Ala, His-E7→Leu, His-E7→Trp, Val-E11→Phe, and Val E11→Trp for alpha and/or beta globin.
 22. A mutant recombinant hemoglobin according to claim 7 wherein one or more of the mutations are selected from the group consisting of Leu-B10→Trp, Leu-B10→Phe, Leu-B10→Val, Leu-B10→Ile, Leu-B10→Ala, His-E7→Leu, His-E7→Trp, Val-E11→Phe, and Val E11→Trp for alpha and/or beta globin.
 23. A mutant recombinant hemoglobin according to claim 8 wherein one or more of the mutations are selected from the group consisting of Leu-B10→Trp, Leu-B10→Phe, Leu-B10→Val, Leu-B10→Ile, Leu-B10→Ala, His-E7→Leu, His-E7→Trp, Val-E11→Phe, and Val E11→Trp for alpha and/or beta globin.
 24. A pharmaceutical composition comprising a mutant recombinant hemoglobin according to claim
 1. 25. A hemoglobin solution comprising a mutant recombinant hemoglobin according to claim
 1. 